International Journal of Organ Transplantation Medicine Antioxidant Effects of Bone Marrow Mesenchymal Stem Cell against Carbon Tetrachloride-induced Oxidative Damage in Rat Livers

Background: Liver fibrosis results from excessive accumulation of extracellular matrix, which affects liver function over time and leads to its failure. In the past, liver transplant was thought to be the only treatment for end-stage liver disease, but due to the shortage of proper donors other medical treatments have been taken into consideration.


INTRODUCTION
L iver transplantation is the most important treatment for patients suffering from liver failure, but this method has limitations due to the shortage of appropri-ate donors. Clinical and laboratory studies have considered alternative therapies for these patients. Recently, mesenchymal stem cell (MSC) has been investigated with the prospect of treatment of acute and chronic liver diseases. Some studies provide clinical and experimental evidence suggesting that MSC transplantation can restore the liver function in acute and chronic damages [1,2]. Other studies have concentrated on hematopoietic cells and stem cells derived from the bone marrow (BM-MSC), which have a great ability for division and implantation in the liver of transplant recipient [3].
In the bone marrow, there are main populations of stem cells including hematopoietic stem cells, MSCs and multipotent adult progenitor cells [4]. A number of studies have proven that under appropriate environmental conditions, cells derived from the bone marrow can differentiate into hepatocytes both in vivo [5,6] and in vitro [7]. We have previously found that BM-MSCs can participate in differentiation of hepatocytes. In vitro differentiated MSCs using IGF-I are able to display advanced liver metabolic functions supporting the possibility to use them as potential alternatives to primary hepatocytes [8].
Administration of MSC can decrease the injury in the liver, lungs and heart by reducing inflammation, collagen deposition and rearrangement [9]. Sakaida, et al [10], and Fang, et al [9], reported that bone marrow cells or MSCs can reduce hepatic fibrosis caused by carbon tetrachloride (CCl 4 ) in mice. However, the reversal of fibrosis by MSC is not completely known so far.
Stem cells have the ability not only to implant in the target tissues, but also to secrete many factors that could change or improve the function of the damaged tissue [11]. Given that after transplantation of the damaged tissues or organs, the stem cells are influenced by several factors such as inflammatory cytokines [12,13], and migration to the target organs after a successful transplantation; the cells can activate processes that lead to reconstruction of the damaged cells and tissues [14,15]. CCl 4 is a more efficient hepatotoxic substance, the toxicity of which is based on the change in its bio-structure into two free radicals and plays an important role in hepatotoxicity, tissue damage and cell death [16]. Oxidative stress contributes to the pathogenesis of various diseases. Tightly regulated defense systems such as glutathione production have evolved to combat these stresses.
There are many controversies on the main mechanisms through which cell-based therapy affects liver tissue repair. The possibility of using human MSCs to repair liver damage has not yet been evaluated. In this study, we investigated the therapeutic use of MSC transplantation on CCl 4 -induced liver failure in rats.

Isolation of human MSCs (hMSCs)
Human MSCs (hMSCs) were isolated using a method previously described [17]. Cells from human bone marrow (BM) were taken from several people from the posterior iliac crest bone by aspiration in Namazi Hospital. Then, they were mixed with complete medium (ratio of 1:1) and mononuclear cells were gently placed in a separate Falcon using density gradient method (Percoll, 1.073 g/mL). The mononuclear cells were then isolated from the BM by centrifuging at 2000 rpm for 20 min at 21 °C and were cultured in complete medium (Dulbecco's modified Eagle's medium [DMEM, Gibco/BRL]) with fetal bovine serum (10%). The cells were then incubated at 37 °C in a medium containing 5% humidity and CO 2 for 12-14 days; during this period, they began to form colonies. Once the colonies developed (filling 80%-90%), the media were washed twice in PBS and the cells were trypsinized with 25% trypsin in 1 mM EDTA (Gibco/BRL) for five minutes at 37 °C. After centrifugation, the cells were suspended in supplemented medium with serum and incubated in a 25-cm 2 flask. Culture media were prepared for the first passage [18]. MSCs were identified in the culture medium by sticking and spindle deformation [19].
At each passage, the cells were counted and Antioxidant effects of BM-MSC against CCl 4 -induced hepatotoxicity analyzed for viability by trypan blue staining analysis. Flow cytometric analysis and functional ability of differentiation into osteocyte and adipocyte was achieved in response to specific culture conditions. Each experiment described here was replicated thrice.

Preparation of animals and experimental groups
Twenty-one adult male Wistar rats weighing 250-300 g were purchased from Razi Institute for Serum and Vaccine and categorized into three groups (7 rats in each group). Group 1 rats (negative control) did not receive any CCl 4 ; they only received olive oil intraperitoneally twice per week for eight weeks. Group 2 rats (positive control) received CCl 4 diluted 1:1 in olive oil intraperitoneally (1 mL/ kg) twice per week for eight weeks. Group 3 rats, in addition to receiving CCl 4 intraperito-neally (1 mL/kg), received the prepared cells (1×10 6 cells in 1 mL PBS) by insulin syringe into several lobes of the liver in a completely sterile environment in the fourth week after CCl 4 injection. After the eighth week of CCl 4 injection, the rats were anesthetized using thiopental (50 mL/kg). Their blood was collected for providing serum and their liver was dried after being washed with normal saline. Segments of their dried liver were collected for assessment of the oxidative stress and the rest was fixed in formalin (10%). Histological sections (5 μm) of the liver-lobes were obtained, stained with hematoxylin, eosin and Masson's trichrome stain, and transported to the lab.

Histopathological studies
A segment of the liver was excised from the animals and fixed in 10% formalin for at least 24 hours. Then, the paraffin sections were prepared and cut into 5-µm sections by a rotary microtome. The sections were stained with hematoxylin-eosin and studied for histopathological changes, ie, necrosis, fatty changes, ballooning degeneration, and inflammation. Histological damages were scored-0: "absent;" +: "mild;" ++: "moderate;" and +++: "severe." Measurement of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin Biocon standard kits and DAX-48 autoanalyzer were used to measure serum ALT, AST, and albumin, according to Wilkinson, et al, and Bessay, et al's method [20,21].

Determination of lipid peroxidation
The extent of lipid peroxidation was assessed by measuring the amount of thiobarbituric acid-reactive substances (TBARs). In brief, 500 mg of the liver tissue was gently minced in 4.5 mL of 0.25 M sucrose. The minced tissue was gently homogenized and then centrifuged at 2000 rpm for 30 min. Afterwards, 0.1 mL of the supernatant was treated with a buffer containing 0.75 mL of thiobarbituric acid (0.8%, w/v), 0.75 mL of 20% acetic acid (pH 3.5) and 0.1 mL of sodium dodecylsulfate (8.1%, w/v). The solution was mixed up with 2 mL of distilled water and heated in a boiling water bath for 60 min. The absorbance was measured at 532 nm by a Beckman DU-7 spectrophotometer [22].

GSH determination
Glutathione reductase 5, 50-dithiobis-2 ni-trobenzoic acid (DTNB) recycling procedure [23] was used to determine the reduced glutathione. In brief, 100 mg of the liver tissue was homogenized in a buffer containing EDTA (0.2 M) to obtain 4% (w/v) whole homogenate. Then, 1.5 mL of the suspension was taken and mixed with a buffer containing 2.5 mL distilled water and 0.5 mL of 50% TCA. The mixture was then centrifuged at 3000 rpm for 15 min, and 1 mL of the supernatant mixed with 1 mL of tris buffer (0.4 M, pH 8.9) and 0.1 mL of DTNB (0.01 M). The absorbance was measured after 5 min at 412 nm using a Beckman DU-7 spectrophotometer [24].

Statistical analysis
The data were analyzed by Student's t test and one-way ANOVA, followed by Graphpad Prism 5. A p value <0.05 was considered statistically significant.

RESULTS
Histological studies of the liver sections from control rats showed normal architecture, characterized by polyhedral shaped hepatocytes with small uniform nuclei. Hepatocytes were arranged in well-organized hepatic cords and separated by narrow blood sinusoids. Hepatic sections, followed by CCl 4 treatment, showed typical CCl 4 -induced hepatic injury (Fig 1B). The sections revealed extremely vacuolated hepatocytes (apoptotic necrotic cells and a lot of foamy cells) adjacent to the central vein (arrows and arrowhead, Fig 1B), indicating chronic CCl 4 -induced hepatotoxicity (with  Table 1). Hepatocytes were significantly protected from CCl 4 -induced toxicity after MSC infusion (Fig 1C), which demonstrated a normal appearance.
The section from liver in Figure 1 shows severe necrosis in the central vein of the rats treated with CCl 4 compared to the control group (with +++ grade, Table 1); there was no necrosis in the CCl 4 + MSC treated group (with 0 grade, Table 1). Liver lobules in the CCl 4 + MSC treated group had a relatively normal appearance, compared to the CCl 4 group with no fatty changes (with 0 grade, Table 1 and Fig 1C). The effects of MSCs on reducing necrosis (with + grade, Table 1) surrounding the central vein area (Fig 1C), and the absence of swollen hepatocytes were clearly shown in the H&E sections.
Histopathological examination of the liver revealed that MSC has anti-fibrosis effects by reducing the amount of collagen which is shown by Masson's trichrome stain compared to the group receiving CCl 4 , which showed significant periportal fibrosis (Fig 2D).
The results showed slight increase in the serum albumin levels in the group receiving CCl 4 + MSC compared to the group receiving CCl 4 alone (Fig 2A). Other liver enzymes also showed significant changes. For instance, AST and ALT in the group treated with CCl 4 + MSC were significantly (p<0.01) lower compared to the group receiving CCl 4 alone (Figs 2B and 2C).
Lipid peroxidation in the liver showed a significant increase in the CCl 4 group compared to the control group (p<0.01); in the CCl 4 + MSC treated group, a significant reduction was observed in lipid peroxidation levels (p<0.01) ( Table 2).
Glutathione (GSH) was also assessed as an indicator of hepatic antioxidant enzymes. A sig- nificant increase was observed in GSH levels in CCl 4 + MSC treated group compared with the CCl 4 group (p<0.05). Moreover, a significant increase was observed in GSH levels in CCl 4 + MSC treated group compared with the control group (p<0.05) ( Table 2).

DISCUSSION
Rats treated with CCl 4 are usually used as an in vivo model for studies on liver damage. CCl 4 usually produces free radicals that trigger a cascade of reactions leading to liver fibrosis. CCl 4 is converted to free radicals by cytochrome P450 which exerts its effects on the liver through lipid peroxidation [25,26].
Oxidative stress is a pathogenic mechanism in the initiation and progression of liver damage involved in many liver disorders. Cell damage occurs when reactive oxygen species concentration increases in the liver. The use of antioxidants reduces the amount of these free radicals [27] .
Glutathione (l-γ-glutamyl-l-cysteinyl-glycine), which is present in all mammalian tissues, especially the liver, provides the reduction capacity for most reactions and plays a very important role in the detoxification of hydrogen peroxide, other peroxides and free radicals [28] .
In the present study, the hepatic content of GSH was found to be decreased significantly in the CCl 4 intoxicated rats compared to the controls. Table 2 shows that MSC-based therapy significantly inhibited the CCl 4 -induced decrease of hepatic GSH content, and it was significantly increased in the MSC-treated group. The resulting hepatocellular toxicity has been demonstrated in numerous studies as reflected by increased liver enzymes.
Because the liver is considered the main organ for biological degradation and subsequently detoxification of harmful substances, it contains important enzymes for their biological actions. When CCl 4 is administered to rats, the activity of AST and ALT in plasma rises significantly with necrosis of and lipid accumulation in hepatocyte [29]. Both enzymes are indicators of liver injury. ALT is more sensitive to acute liver injury, whereas AST is more sensitive to chronic injury [30]. In this study, we investigated alteration of liver enzymes after transplantation of MSCs into the rat liver injured with CCl 4 . MSCs transplantation restored the increase of liver enzymes as well as the up regulation of albumin.
Recovery of liver function after MSCs transplantation was also examined by histological changes. MSCs reversed the hepatic necrosis, fatty changes, and inflammation. However, the mechanisms by which MSCs repair liver damage remain unclear. HGF is one of the factors produced by the MSC and its beneficial effects include mitogens, morphogens, and anti-tumor activities [31]. Specifically, it was determined that HGF exerts beneficial effects on liver damage. MSC transplantation regenerates the reduction in hepatic protective genes and shows its effects by reducing LP, as determined by the amount of GSH in the liver. It was also determined that in ischemic/ perfusion damage to liver, MSC transplanta- tion leads to suppression of oxidative stress and reduction in the amount of apoptosis in rats [32].
We have shown that MSC transplantation may protect liver injury by altering the oxidative effect of CCl 4 by increasing GSH content of the liver. In conclusion, we demonstrated the protective effect of MSCs, and suggest that bone marrow-derived MSCs could be a therapeutic approach for liver damage, particularly for those due to oxidative stress.